Membrane Lipid Domains Modulate HCN Channels in Nociceptor DRG Neurons.

Cell membranes consist of heterogeneous lipid domains that influence key cellular processes, including signal transduction, endocytosis, and electrical excitability. Using FRET-based fluorescent assays and fluorescence lifetime imaging microscopy (FLIM), we found that the dimension of cholesterol-enriched ordered membrane domains (OMD) varies considerably, depending on specific cell types. The size of OMDs is also dependent on cholesterol levels and the structure of lipid tails. Particularly, nociceptor dorsal root ganglion (DRG) neurons exhibit large OMDs. Disruption of OMDs potentiated action potential firing in nociceptor DRG neurons and facilitated opening of native hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. This increased neuronal firing could be partially due to an increased open probability of HCN channels. In animal models of neuropathic pain, we observed shrunken OMDs and relocalization of HCN channels from OMDs to disordered lipid domains. The gating effect on HCN channels was likely a result of direct modulation of the voltage sensor by OMDs. These findings suggest that disturbances in lipid domains play a role in regulating HCN channels within nociceptor DRG neurons, influencing pain modulation.

Direct regulation of the voltage sensor of HCN channels by membrane lipid compartmentalization.

Ion channels function within a membrane environment characterized by dynamic lipid compartmentalization. Limited knowledge exists regarding the response of voltage-gated ion channels to transmembrane potential within distinct membrane compartments. By leveraging fluorescence lifetime imaging microscopy (FLIM) and Förster resonance energy transfer (FRET), we visualized the localization of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in membrane domains. HCN4 exhibits a greater propensity for incorporation into ordered lipid domains compared to HCN1. To investigate the conformational changes of the S4 helix voltage sensor of HCN channels, we used dual stop-codon suppression to incorporate different noncanonical amino acids, orthogonal click chemistry for site-specific fluorescence labeling, and transition metal FLIM-FRET. Remarkably, altered FRET levels were observed between VSD sites within HCN channels upon disruption of membrane domains. We propose that the voltage-sensor rearrangements, directly influenced by membrane lipid domains, can explain the heightened activity of pacemaker HCN channels when localized in cholesterol-poor, disordered lipid domains, leading to membrane hyperexcitability and diseases.